1Department of Neurosurgery, 2Graduate Program
in Neuroscience, 3Stem Cell Institute, Division of Hematology,
Oncology and Transplantation, Department of Medicine, and 4Department
of Genetics, Cell Biology and Development, University of Minnesota Medical
School, Minneapolis, MN 55455

Previously we reported the characterization of multipotent adult progenitor
cells (MAPCs) isolated from the bone marrow of rodents. In that study,
single murine MAPCs derived from ROSA-26, b-galactosidase
(b-Gal)-positive transgenic mice were injected
into E3.5 C57/Bl6 mouse blastocysts. The resultant chimeric blastocysts
were then implanted into pseudopregnant females and were allowed to develop
naturally through birth and into adulthood. Chimeric mice were sacrificed
6 to 20 weeks after birth, and were processed for histological analysis.
b-Galactosidase activity was identified in all
organs and tissues examined, and tissue-specific differentiation and engraftment
was confirmed by colabeling with antibodies that recognize b-Gal
and tissue-specific markers. In the present study we have examined neural
engraftment derived from the clonal expansion of a single MAPC during rodent
development, and characterized the neural phenotype of MAPCs in the resultant
chimeric animals. Donor cell-derived b-Gal activity
was evident throughout the brain. Double and triple immunofluorescent labeling
studies revealed MAPC-derived neurons (NeuN/b-Gal)
and astrocytes (GFAP/b-Gal) in the cortex, striatum,
medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus.
More specifically, donor-derived neurons contributed to each of the cellular
layers of the cortex; the pyramidal and granule cell layers, as well as
the hilus, of the hippocampus; Purkinje and granule cell layers in the
cerebellum; and GABAergic cells in the caudate and putamen. This study
characterizes the potential for MAPCs to differentiate into specific neuronal
and glial phenotypes, and to integrate normally during development, after
implantation into blastocysts, and provides additional evidence that MAPCs
exhibit properties similar to embryonic stem cells.

Embryonic neural precursors (ENPs), also termed neural stem cells or
"neurospheres," are an attractive potential source of tissue for neural
transplantation, because of their capacity to expand in number in vitro
while retaining the ability to develop into the major phenotypes of the
CNS. ENPs are isolated from the developing brain and proliferate in the
presence of mitogens such as FGF-2 and EGF. Subsequent withdrawal of these
mitogens and exposure to a suitable substrate results in differentiation
into the major cell types of the CNS. As well as its role in precursor
cell expansion, FGF-2 also plays a key role in the division of astrocytes,
and in neuronal differentiation. Thus, it is important to establish the
optimal concentrations of this factor for expansion and differentiation
of neuronal phenotypes. Here we explore the effect of FGF-2 concentrations
ranging from 1 to 20 ng/ml on the expansion and differentiation capacity
of ENPs isolated from the cortex and striatum of E14 mice. ENP expansion
was seen under all conditions, but was greatest at 10 and 20 ng/ml and
least at 1 ng/ml. The numbers of neurons (as a proportion of total cell
number) differentiating from these ENP populations appeared to be greatest
at 1 ng/ml. However, once adjustments were made for the amount of expansion
at each dose, final neuronal yield was maximum at the highest concentration
of FGF-2 used (20 ng/ml).

Poor survival and differentiation of grafted dopamine neurons limits
the application of clinical transplantation in Parkinson's disease. The
survival of grafted dopamine neurons is only improved by a factor of 2-3
by adding neuroprotectants during tissue preparation. We used dye exclusion
cell viability and electron microscopy to investigate the effects of the
caspase inhibitor ac-YVAD-cmk and the lazaroid tirilazad mesylate on ultrastructural
changes in dissociated embryonic mesencephalic cells. In addition, we examined
whether the neuroprotectants selectively counteracted specific signs of
neurodegeneration. Cell viability decreased significantly over time in
both control and treated cell suspensions, but the number of viable cells
remaining was significantly higher in tirilazad mesylate-treated cell suspensions.
In control samples, the proportion of cells with an ultrastructure consistent
with healthy cells decreased from 70%, immediately after dissociation,
to 30% after 8 h of incubation. Similar changes were also observed in cell
suspensions treated with neuroprotectants. Thus, the neuroprotectants examined
did not block the development of specific morphological signs of neurodegeneration.
However, when also taking into account that dead cells lysed and disappeared
from each cell suspension with time, we found that the total number of
remaining viable cells with healthy nuclear chromatin or intact membrane
integrity was significantly higher in the tirilazad mesylate-treated group.
The results indicate that tirilazad mesylate protects only a small subpopulation
of embryonic mesencephalic cells from degeneration induced by mechanical
trauma during tissue dissection and dissociation.

The purpose of this study was to investigate the influence of fetal
lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal
ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and
LGE plated as single or cocultures were employed. Survival time was 3-21
days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed
using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition,
cultures were labeled with the TUNEL technique and with antibodies directed
against neurofilament (NF) in order to study apoptosis and retraction of
nerve fibers, respectively. The results revealed two morphologically different
types of TH-positive outgrowth growing into the substrate. The initially
formed TH-positive outgrowth radiated continuously without changing direction,
while a second wave of TH-positive outgrowth became obvious when the initial
growth already had reached a distance of approximately 1000 mm.
The second wave of TH-positive outgrowth radiated from the tissue, but
at a certain distance changed direction and formed a network surrounding
the culture. The initially formed TH-positive growth was not associated
with the presence of S100-positive astrocytes and avoided to grow into
the LGE. At longer time points the first wave of TH-positive nerve fibers
appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures
these long distance growing fibers had disappeared at 21 days in vitro.
The second wave of TH-positive nerve fibers was growing onto a layer of
glia and never reached the distance of the first wave. LGE became innervated
by TH-positive fibers at the time point for when the second wave of TH-positive
growth had been initiated, and the innervation appeared in TH-dense patches
that also showed a high density of S100-positive astrocytes. Significantly
increased TUNEL activity within LGE portion of cocultures was observed
when TH-positive fibers entered the LGE and formed patches. In conclusion,
two morphologically different types of TH-positive outgrowth were found
and the initially formed fibers neither targeted the LGE nor were they
guided by glial cells, but their potential to grow for long distances was
high.

The Role of Pretraining on Skilled Forelimb Use
in an Animal Model of Huntington's Disease

R. A. Fricker-Gates, R. Smith, J. Muhith, and S. B. Dunnett

Brain Repair Group, School of Biosciences, Cardiff University, UK

After a unilateral striatal lesion, animals have generally been seen
to have a bilateral impairment in paw reaching, with the contralateral
paw being more affected. However, most studies to date have not used a
pretraining paradigm to assess maximal capacity for paw reaching, to compare
with any lesion-induced loss. This study compared animals that were pretrained
with naive animals in their ability to paw reach after a striatal lesion,
to address the role of the striatum in either acquisition or execution
of this motor task. All lesioned animals showed a significant decrease
in reaching ability with their contralateral paw compared with the ipsilateral
paw. Pretrained lesioned animals showed a clear lesion deficit with the
contralateral paw immediately after lesion, and no impairment whatsoever
with the ipsilateral paw. Naive lesioned animals showed delayed acquisition
of the task with both paws, possibly due to postural deficits, and a lasting
deficit on the contralateral side. The variability of performance between
animals was higher in the naive lesioned group. These results suggest that
animals should be pretrained on the staircase task prior to lesion to enable
maximum sensitivity in detecting both loss and recovery of function of
skilled forelimb use.

Basic fibroblast growth factor (FGF-2) has been shown to enhance the
survival and neurite extension of various types of neurons including spinal
ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated
following peripheral nerve lesion in ganglia and at the lesion site. FGF-2
protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and
differentially regulated after nerve injury. In the rat we analyzed the
regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms
(21/23 kDa) to support the regeneration of the axotomized adult sciatic
nerve across long gaps. The nerve stumps were inserted into the opposite
ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone
tubes were filled with Matrigel or a mixture of Schwann cells (SC) and
Matrigel. SC were prepared from newborn rats and transfected to overexpress
HMW FGF-2. Four weeks after the operation procedure, channels were analyzed
with regard to tissue cables bridging both nerve stumps and myelinated
axons distal to the original proximal nerve stump. Peripheral nerves interposed
with HMW Schwann cells displayed significantly enhanced nerve regeneration,
with the greatest number of tissue cables containing myelinated axons and
the highest number of myelinated axons. These results suggest that a cellular
substrate together with a source of a trophic factor could be a promising
tool to promote nerve regeneration and, therefore, become useful also for
a clinical approach to repair long gaps.

1Kentucky Spinal Cord Injury Research Center and Departments
of 2Neurological Surgery and 3Anatomical Sciences
and Neurobiology, University of Louisville School of Medicine, Louisville,
KY 40202
4Department of Physiology, University of Puerto Rico School
of Medicine, San Juan, PR 00936

Eph receptors and ligands represent two families of proteins that control
axonal guidance during development. Recent work has shown that several
Eph receptors are expressed postnatally. Because the Eph molecules represent
a class of axon guidance molecules that are mainly inhibitory to axonal
growth, we investigated whether EphB3 expression was upregulated in both
spinal cord and four supraspinal nuclei (locus coeruleus, vestibular, raphe
pallidus, and red) 1 week after a complete spinal cord thoracic transection.
Injured rats had a significant increase in EphB3 mRNA and protein expression
in the spinal cord. The increased EphB3 expression was colocalized with
GFAP staining and indicated that astrocytes play a role in EphB3 expression
after spinal cord injury. No change in EphB3 expression was seen in supraspinal
brain nuclei, which further demonstrated that changes in expression were
due to changes in the local microenvironment at the injury site. The expression
of EphB3 was colocalized to regions of the CNS that had a high level of
EphB3 binding ligands. These data indicate upregulation of EphB3 expression
after injury may also contribute to an environment in the spinal cord that
is inhibitory to axonal regeneration.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor
for noradrenergic (NE) neurons of the pontine nucleus locus coeruleus (LC).
Decreased function of the LC-NE neurons has been found during normal aging
and in neurodegenerative disorders. We have previously shown that GDNF
participates in the differentiation of LC-NE neurons during development.
However, the continued role of GDNF for LC-NE neurons during maturation
and aging has not been addressed. We examined alterations in aged mice
that were heterozygous for the GDNF gene (Gdnf+/-). Wild-type
(Gdnf+/+) and Gdnf+/- mice (18 months
old) were tested for locomotor activity and brain tissues were collected
for measuring norepinephrine levels and uptake, as well as for morphological
analysis. Spontaneous locomotion was reduced in Gdnf+/-
mice in comparison with Gdnf+/+ mice. The reduced locomotor
activity of Gdnf+/- mice was accompanied by reductions
in NE transporter activity in the cerebellum and brain stem as well as
decreased norepinephrine tissue levels in the LC. Tyrosine hydroxylase
(TH) immunostaining demonstrated morphological alterations of LC-NE cell
bodies and abnormal TH-positive fibers in the hippocampus, cerebellum,
and frontal cortex of Gdnf+/- mice. These findings suggest
that the LC-NE system of Gdnf+/- mice is impaired and
suggest that GDNF plays an important role in continued maintenance of this
neuronal system throughout life.